Membrane-associated Protein Kinase Research Articles

A plasma membrane-associated form of the androgen receptor enhances nuclear androgen signaling in osteoblasts and prostate cancer cells.

Androgen binding to the androgen receptor (AR) in the cytoplasm induces the AR to translocate to the nucleus, where it regulates the expression of target genes. Here, we found that androgens rapidly activated a plasma membrane-associated signaling node that enhanced nuclear AR functions. In murine primary osteoblasts, dihydrotestosterone (DHT) binding to a membrane-associated form of AR stimulated plasma membrane-associated protein kinase G type 2 (PKG2), leading to the activation of multiple kinases, including ERK. Phosphorylation of AR at Ser515 by ERK increased the nuclear accumulation and binding of AR to the promoter of Ctnnb1, which encodes the transcription factor β-catenin. In male mouse osteoblasts and human prostate cancer cells, DHT induced the expression of Ctnnb1 and CTNN1B, respectively, as well as β-catenin target genes, stimulating the proliferation, survival, and differentiation of osteoblasts and the proliferation of prostate cancer cells in a PKG2-dependent fashion. Because β-catenin is a master regulator of skeletal homeostasis, these results explain the reported male-specific osteoporotic phenotype of mice lacking PKG2 in osteoblasts and imply that PKG2-dependent AR signaling is essential for maintaining bone mass in vivo. Our results suggest that widely used pharmacological PKG activators, such as sildenafil, could be beneficial for male and estrogen-deficient female patients with osteoporosis but detrimental in patients with prostate cancer.

The Role of MARCKS in Metastasis and Treatment Resistance of Solid Tumors.

Simple SummaryCancer metastasis is a critical event in the progression of solid tumors and is invariably associated with adverse outcomes and mortality. Understanding novel mechanisms or molecules that promote cancer metastasis will facilitate the development of new strategies for cancer treatment. Recently, MARCKS has been studied extensively in several cancers and has been implicated in tumor progression and metastasis. This review summarizes recent advances in the understanding of MARCKS on cancer metastasis, stemness, and therapeutic resistance and provides prospects on targeting MARCKS therapeutically. Specifically, we review the molecular mechanisms and multiple signaling pathways by which MARCKS contributes to the progression and metastasis in solid tumors.The myristoylated alanine-rich C-kinase substrate (MARCKS) is a membrane-associated protein kinase C (PKC) substrate ubiquitously expressed in eukaryotic cells. MARCKS plays important roles in multiple cellular processes, including cell adhesion and motility, mucin secretion, exocytosis, and inflammatory response. Aberrant MARCKS signaling has been observed in the development and progression of multiple cancer types. In addition, MARCKS facilitates cancer metastasis through modulating cancer cell migration and invasion. Moreover, MARCKS contributes to treatment resistance, likely by promoting cancer stem cell renewal as well as immunosuppression. In this review, we describe MARCKS protein structure, cellular localization, and biological functions. We then discuss the role of MARCKS in cancer metastasis as well as its mechanisms of action in solid tumors. Finally, we review recent advances in targeting MARCKS as a new therapeutic strategy in cancer management.

Abstract 3757: Examination of the epidermal growth factor 3 (HER3) dysregulations in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide with a particularly high mortality rate. Treatment options for HCC are limited. Curative treatments including surgical resection and liver transplantation are only applicable to early diagnosed patients. For advanced HCC patients, the currently available first-line molecularly targeted medications are suffering from low rates of response and high inter-patients variability providing limited survival benefits. Receptor tyrosine kinases (RTKs) are plasma membrane-associated protein kinases which control the activation of intracellular signal transductions in response to the extracellular stimuli. Activation of specific RTKs has been implicated in various kinds of human cancers, and pharmacological inhibition of cancer associated RTKs is one of the key strategies of modern targeted therapy. However, a comprehensive and systematic assessment of the RTKs dysregulations in terms of expression and mutation in HCC is still much awaited. In this study, RNA-sequencing analysis was performed in paired HCC tissues to examine the expression profiles of 58 RTKs. Also, the mutational status of these RTKs in HCC tissues was examined by targeted DNA-sequencing analysis. Our systemic analyses have revealed that Epidermal Growth Factor Receptor 3 (HER3) was significantly upregulated and recurrently mutated in HCC. Independent qPCR analysis for HER3 in 101 paired HBV-associated HCC tumor and non-tumorous samples validated that HER3 was upregulated in HCC. More importantly, upregulation of HER3 was found to be significantly correlated with the presence of metastatic features of HCC including tumor microsatellite formation (p=0.015) and venous invasion (p=0.001). Neuregulin-1 (NRG-1) is the best-known ligand for HER3. Stimulation of HER3 activation by the ligand NRG-1 could induce HER3 phosphorylation, and activate the downstream PI3K/AKT and MAPK signaling pathways, and signficantly promoted the proliferation and migration of HCC cells. Conversely, CRISPR/Cas9-mediated HER3 knockout in Huh7 and PLC cells significantly reduced the NRG-1 induced functional changes. Consistently, our preliminary results demonstrated that HER3 knockout Huh7 cells forms relatively smaller tumors in a subcutaneous tumor xenograft model in vivo. Together, our results demonstrated that HER3 dysregulation plays a supporting role in HCC development. Targeting HER3 may potentially derive new therapeutic strategy for treatment of HCC. Citation Format: Sheung In Ming, Lo Kong Chan, Daniel W.H. Ho, Irene O.L. Ng. Examination of the epidermal growth factor 3 (HER3) dysregulations in hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3757.

The Effect of Lithium Binding on Secondary and Tertiary Structure, Hydrophobicity, Thermodynamics, and Interactions with Interacting Partners of Dream

Li+ is widely used for the treatment of manic-depressive illness. Studies on neuronal calcium sensor protein1 (NCS-1) have found that Li+ can inhibit the interaction between NCS-1 and inositol 1,4,5-triphosphate receptor protein, alleviating hyperarousal of insomnia patient. Li+ can have neuroprotective or neurotoxic effects depending on its concentration. In a rat model, chronic exposure to Li+ has shown a significant decrease in membrane-associated protein kinase C (PKC) in the hippocampus, suggesting potential clinical relevance. Here, we show that Li+ binds to a neuronal calcium sensor protein named Downstream Element Antagonist Modulator (DREAM), a protein homologous to NCS-1 and expressed in the hippocampus region of the brain. Li+ association results in a decrease in the emission intensity of tryptophan, suggesting rearrangements of the tertiary structure of the protein. Li+ binding also exposes hydrophobic cavity of the protein as evidenced by binding of the hydrophobic molecule 1,8-ANS. CD data reveal that Li+ binding alters the protein secondary structure. Tryptophan emission and CD data are further supported by steady-state lifetime data. Thermodynamic parameters for Li+ association to DREAM have been obtained through isothermal titration calorimetry (ITC) measurements. Li+-bound DREAM interactions with binding partners have been probed by titrating FITC-tagged presenilin-1 and potassium channel against Li+-bound DREAM. Findings of this project suggest potentials for the Li+-based compounds in the animal model to investigate whether acute and chronic exposure to Li+ has any effect on the expression level of DREAM and whether Li+-based compounds influence interactions between DREAM and intracellular partners.

The Stress-Sensing TORC2 Complex Activates Yeast AGC-Family Protein Kinase Ypk1 at Multiple Novel Sites

Yeast (Saccharomyces cerevisiae) target of rapamycin (TOR) complex 2 (TORC2) is a multi-subunit plasma membrane-associated protein kinase and vital growth regulator. Its essential functions are exerted via phosphorylation and stimulation of downstream protein kinase Ypk1 (and its paralog Ypk2). Ypk1 phosphorylates multiple substrates to regulate plasma membrane lipid and protein composition. Ypk1 function requires phosphorylation of Thr504 in its activation loop by eisosome-associated Pkh1 (and its paralog Pkh2). For cell survival under certain stresses, however, Ypk1 activity requires further stimulation by TORC2-mediated phosphorylation at C-terminal sites, dubbed the “turn” (Ser644) and “hydrophobic” (Thr662) motifs. Here we show that four additional C-terminal sites are phosphorylated in a TORC2-dependent manner, collectively defining a minimal consensus. We found that the newly identified sites are as important for Ypk1 activity, stability, and biological function as Ser644 and Thr662. Ala substitutions at the four new sites abrogated the ability of Ypk1 to rescue the phenotypes of Ypk1 deficiency, whereas Glu substitutions had no ill effect. Combining the Ala substitutions with an N-terminal mutation (D242A), which has been demonstrated to bypass the need for TORC2-mediated phosphorylation, restored the ability to complement a Ypk1-deficient cell. These findings provide new insights about the molecular basis for TORC2-dependent activation of Ypk1.

Cluster Differentiating 36 (CD36) Deficiency Attenuates Obesity-Associated Oxidative Stress in the Heart.

RationaleObesity is often associated with a state of oxidative stress and increased lipid deposition in the heart. More importantly, obesity increases lipid influx into the heart and induces excessive production of reactive oxygen species (ROS) leading to cell toxicity and metabolic dysfunction. Cluster differentiating 36 (CD36) protein is highly expressed in the heart and regulates lipid utilization but its role in obesity-associated oxidative stress is still not clear.ObjectiveThe aim of this study was to determine the impact of CD36 deficiency on cardiac steatosis, oxidative stress and lipotoxicity associated with obesity.Methods and ResultsStudies were conducted in control (Lean), obese leptin-deficient (Lepob/ob) and leptin-CD36 double null (Lepob/obCD36-/-) mice. Compared to lean mice, cardiac steatosis, and fatty acid (FA) uptake and oxidation were increased in Lepob/ob mice, while glucose uptake and oxidation was reduced. Moreover, insulin resistance, oxidative stress markers and NADPH oxidase-dependent ROS production were markedly enhanced. This was associated with the induction of NADPH oxidase expression, and increased membrane-associated p47phox, p67phox and protein kinase C. Silencing CD36 in Lepob/ob mice prevented cardiac steatosis, increased insulin sensitivity and glucose utilization, but reduced FA uptake and oxidation. Moreover, CD36 deficiency reduced NADPH oxidase activity and decreased NADPH oxidase-dependent ROS production. In isolated cardiomyocytes, CD36 deficiency reduced palmitate-induced ROS production and normalized NADPH oxidase activity.ConclusionsCD36 deficiency prevented obesity-associated cardiac steatosis and insulin resistance, and reduced NADPH oxidase-dependent ROS production. The study demonstrates that CD36 regulates NADPH oxidase activity and mediates FA-induced oxidative stress.

G protein‐coupled Receptor 30 (GPR30) PDZ‐dependently and Constitutively Increases ERK1/2 Signaling Through Calcineurin and Kinase Suppressor of Ras 2 (KSR2)

The objective of the present study was to map the mechanism whereby G protein‐coupled receptor 30 (GPR30), also called G protein‐coupled estrogen receptor (GPER), stimulates extracellular signal‐regulated kinase (ERK)1/2 signaling. GPR30 plays important roles in cancer and cardiometabolic regulation. We showed recently that GPR30 forms a plasma membrane complex through its C‐terminal type I PSD‐95/Discs‐large/ZO‐1 homology (PDZ) motif with a membrane‐associated guanylate kinase (MAGUK) and protein kinase A (PKA)‐anchoring protein 5 (AKAP5), and AKAP5‐anchored PKA regulatory subunit RII suppresses receptor endocytosis and enables the receptor to constitutively inhibit cAMP production. Here, we investigated if this PDZ‐dependent GPR30 complex also regulates ERK1/2 signaling. To do so, human and mouse GPR30 were ectopically expressed in HEK293 cells and MDCK cells, and receptors and effectors were monitored by immunoblotting, immunoprecipitation, confocal immunofluorescence microscopy, split luciferase reporter techniques, and reporter gene assays. We found that GPR30 constitutively increased extracellular signal‐regulated kinase (ERK) 1/2 activity in several cell systems. The response was dependent on an intact receptor PDZ motif. Furthermore, knocking down AKAP5 or inhibiting calcineurin with FK506 inhibited the receptor response. GPR30 PDZ‐dependently inhibited basal NFAT signaling, consistent with the receptor favoring AKAP5‐anchoring of calcineurin. The calcineurin substrate kinase suppressor of Ras 2 (KSR2), a mitogen‐activated protein kinase (MAPK) scaffold, enhanced the GPR30‐promoted response, also dependently on the receptor PDZ motif. GPR30 also PDZ‐dependently favored a membrane KSR2 complex at the expense of the monomeric form. On the other hand, disrupting AKAP5‐PKA RII interaction with St‐Ht31, or inhibiting protein kinase C (PKC) with GF109203X or epidermal growth factor receptor (EGFR) tyrosine kinase with AG1478 had no effect on the GPR30‐stimulated response. FK506 also increased the amount of GPR30 in the plasma membrane, thus acting opposite to St‐Ht31, which increased receptor endocytosis. We conclude that the PDZ‐dependent GPR30 complex with AKAP5 includes calcineurin, which favors GPR30 endocytosis and enables the receptor to constitutively increase ERK1/2 signaling through KSR2.Support or Funding InformationSwedish Cancer Foundation

G protein-coupled Receptor 30 (GPR30) Forms a Plasma Membrane Complex with Membrane-associated Guanylate Kinases (MAGUKs) and Protein Kinase A-anchoring Protein 5 (AKAP5) That Constitutively Inhibits cAMP Production

GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane.

Regulation of Basal Lateral Membrane Mobility and Permeability to Divalent Cations by Membrane Associated-Protein Kinase C

Biological membrane stabilization is essential for maintenance of cellular homeostasis, functionality and appropriate response to various stimuli. Previous studies have showed that accumulation of PKCs in the cell membrane significantly downregulates the membrane fluidity and Ca2+ influxes through the membranes in activated cells. In addition, membrane-inserted form of PKCs has been found in a variety of resting mammalian cells and tissues. This study is aimed to investigate possible role of the endogenous membrane-associated PKCs in the modulation of basal membrane fluidity. Here, we showed that interfering PKC expression by chronic activation of PKC with phorbol myristate acetate (PMA) or shRNA targeting at PKCα lowered the levels of PKCα in cytosol, peripheral membrane and integral membrane pools, while short-term activation of PKC with PMA induced accumulation of PKCα in the membrane pool accompanied by a dramatic decrease in the cytosol fraction. The lateral membrane mobility increased or decreased in accordance with the abundance alterations in the membrane-associated PKCα by these treatments. In addition, membrane permeability to divalent cations including Ca2+, Mn2+ and Ba2+ were also potentiated or abrogated along with the changes in PKC expression on the plasma membrane. Membrane stabilizer ursodeoxycholate abolished both of the enhanced lateral membrane mobility and permeability to divalent cations due to PKCα deficiency, whereas Gö6983, a PKC antagonist, or Gd3+ and 2-aminoethyoxydipheyl borne, two Ca2+ channels blockers, showed no effect, suggesting that this PKC-related regulation is independent of PKC activation or a modulation of specific divalent cation channel. Thus, these data demonstrate that the native membrane-associated PKCα is involved in the maintenance of basal membrane stabilization in resting cells.

New insights on regulation of LMTK2, a membrane kinase integrating pathways central to neurodegeneration

In a short communication in this issue (Manser et al. 2012), Christopher Miller’s group at the Institute of Psychiatry, King’s College London present an elegant and convincing set of experiments using molecular techniques to show that a brain-enriched membrane-associated protein kinase, lemur tyrosine kinase-2 (LMTK2), is directly phosphorylated by the cyclin-dependent kinase-5/p35 and this event is sufficient for LMTK2 to phosphorylate an abundant protein phosphatase, PP1C. LMTK2 has been little studied to date and, despite its name, is a kinase which phosphorylates serine or threonine residues of protein substrates. The paper adds to the evidence that this enzyme is a potentially important mediator positioned to integrate a number of intracellular signalling pathways relevant to neurodegeneration.

Hepatic steatosis by dietary‐conjugated linoleic acid is accompanied by accumulation of diacylglycerol and increased membrane‐associated protein kinase C ε in mice

Conjugated linoleic acid reduces weight gain and adipose mass while inducing liver enlargement, hepatic steatosis, and insulin resistance in mice. The objective of this study was to determine if hepatic steatosis induced by conjugated linoleic acid would predict for hepatic diacylglycerol accumulation, increased membrane-associated protein kinase C ε, and hyperglycemia. Six-wk-old C57Bl/6 male mice were fed a high-saturated fat diet for 3 wk and were then randomized to high-saturated fat diet with or without conjugated linoleic acid (1.5% wt). Following a 6-wk intervention, hepatic triacylglycerol, diacylglycerol, membrane-associated protein kinase C ε, and gluconeogenic gene expression were determined. Fasting glucose was determined at baseline and at the end of the study. Conjugated linoleic acid increased hepatic triacylglycerol and diacylglycerol concentration in association with increased membrane-associated protein kinase C ε. Diacylglycerol concentration proved to be a better predictor than triacylglycerol concentration for the change from baseline in fasting glucose concentration and final insulin concentration. The increase in diacylglycerol concentration was also associated with increased hepatic gluconeogenic gene expression in conjugated linoleic acid-treated animals. Our data suggest that conjugated linoleic acid can initiate the pathophysiology responsible for hepatic insulin resistance. Additional studies are needed to determine if the accumulation of hepatic diacylglycerol by conjugated linoleic acid leads to hepatic insulin resistance.

A protein kinase network regulates the function of aminophospholipid flippases

Limited exposure of aminophospholipids on the outer leaflet of the plasma membrane is a fundamental feature of eukaryotic cells and is maintained by the action of inward-directed P-type ATPases ("flippases"). Yeast S. cerevisiae has five flippases (Dnf1, Dnf2, Dnf3, Drs2, and Neo1), but their regulation is poorly understood. Two paralogous plasma membrane-associated protein kinases, Pkh1 and Pkh2 (orthologs of mammalian PDK1), are required for viability of S. cerevisiae cells because they activate several essential downstream protein kinases by phosphorylating a critical Thr in their activation loops. Two such targets are related protein kinases Ypk1 and Ypk2 (orthologs of mammalian SGK1), which have been implicated in multiple processes, including endocytosis and coupling of membrane expansion to cell wall remodeling, but the downstream effector(s) of these kinases have been elusive. Here we show that a physiologically relevant substrate of Ypk1 is another protein kinase, Fpk1, a known flippase activator. We show that Ypk1 phosphorylates and thereby down-regulates Fpk1, and further that a complex sphingolipid counteracts the down-regulation of Fpk1 by Ypk1. Our findings delineate a unique regulatory mechanism for imposing a balance between sphingolipid content and aminophospholipid asymmetry in eukaryotic plasma membranes.

Oxalate-induced activation of PKC-α and -δ regulates NADPH oxidase-mediated oxidative injury in renal tubular epithelial cells

Oxalate-induced oxidative stress contributes to cell injury and promotes renal deposition of calcium oxalate crystals. However, we do not know how oxalate stimulates reactive oxygen species (ROS) in renal tubular epithelial cells. We investigated the signaling mechanism of oxalate-induced ROS formation in these cells and found that oxalate significantly increased membrane-associated protein kinase C (PKC) activity while at the same time lowering cytosolic PKC activity. Oxalate markedly translocated PKC-alpha and -delta from the cytosol to the cell membrane. Pretreatment of LLC-PK1 cells with specific inhibitors of PKC-alpha or -delta significantly blocked oxalate-induced generation of superoxide and hydrogen peroxide along with NADPH oxidase activity, LDH release, lipid hydroperoxide formation, and apoptosis. The PKC activator PMA mimicked oxalate's effect on oxidative stress in LLC-PK1 cells as well as cytosol-to-membrane translocation of PKC-alpha and -delta. Silencing of PKC-alpha expression by PKC-alpha-specific small interfering RNA significantly attenuated oxalate-induced cell injury by decreasing hydrogen peroxide generation and LDH release. We believe this is the first demonstration that PKC-alpha- and -delta-dependent activation of NADPH oxidase is one of the mechanisms responsible for oxalate-induced oxidative injury in renal tubular epithelial cells. The study suggests that the therapeutic approach might be considered toward attenuating oxalate-induced PKC signaling-mediated oxidative injury in recurrent stone formers.

Adenosine deamination to inosine in isolated basolateral membrane from kidney proximal tubule: Implications for modulation of the membrane-associated protein kinase A

In this work, the metabolism of adenosine by isolated BLM associated-enzymes and the implications of this process for the cAMP-signaling pathway are investigated. Inosine was identified as the major metabolic product, suggesting the presence of adenosine deaminase (ADA) activity in the BLM. This was confirmed by immunoblotting and ADA-specific enzyme assay. Implications for the enzymatic deamination of adenosine on the receptor-modulated cAMP-signaling pathway were also investigated. We observed that inosine induced a 2-fold increase in [ 35S] GTPγS binding to the BLM and it was inhibited by 10 −6 M DPCPX, an A 1 receptor-selective antagonist. Inosine (10 −7 M) inhibited protein kinase A activity in a DPCPX-sensitive manner. Molecular association between ADA and G αi-3 protein-coupled A 1 receptor was demonstrated by co-immunoprecipitation assay. These data show that adenosine is deaminated by A 1 receptor-associated ADA to inosine, which in turn modulates PKA in the BLM through A 1 receptor-mediated inhibition of adenylyl cyclase.

Zeo1, a negative regulator of the cell integrity MAPK pathway, a substrate for the Yck1,2 casein kinase 1 protein kinases

Yeast casein kinase 1 (CK1) proteins Yck1 and 2 are redundant, essential plasma membrane-associated protein kinases in budding yeast. Yck1,2 are required for endocytic trafficking of receptors and permeases, cellular morphogenesis and cytokinesis. Although much is known about Yck1,2 function, it has been difficult to identify substrates using genetic approaches. We used a comparative phosphoproteomic approach to identify Yck1,2 substrates. Protein extracts from wild-type cells and cells with low Yck1,2 activity (yckts cells), were digested and enriched for phosphopeptides. Phosphopeptides were identified by mass spectrometry, and their abundances were compared. One phosphopeptide found to be substantially more abundant in wild-type protein extracts than in yckts extracts is a peptide from Zeo1 that is phosphorylated within a weak CK1 consensus site. Zeo1 was identified as a negative regulator of the cell integrity Pkc1-Slt2 MAP kinase pathway. Zeo1 interacts with Mid2, an upstream regulator of Rho1, and deletion of ZEO1 causes constitutive activation of the Slt2 MAPK. We found that the yckts mutant also shows constitutive activation of Slt2, and yckts genetically interacts with rom2Δ and sac7Δ similar to those for zeo1Δ. We are now testing for in vitro phosphorylation of Zeo1 by Yck2, and are confirming preliminary observations that mutation of the Zeo1 phosphorylation site affects Zeo1 activity in vivo.

Plectin-controlled keratin cytoarchitecture affects MAP kinases involved in cellular stress response and migration

Plectin is a major intermediate filament (IF)–based cytolinker protein that stabilizes cells and tissues mechanically, regulates actin filament dynamics, and serves as a scaffolding platform for signaling molecules. In this study, we show that plectin deficiency is a cause of aberrant keratin cytoskeleton organization caused by a lack of orthogonal IF cross-linking. Keratin networks in plectin-deficient cells were more susceptible to osmotic shock–induced retraction from peripheral areas, and their okadaic acid–induced disruption (paralleled by stress-activated MAP kinase p38 activation) proceeded faster. Basal activities of the MAP kinase Erk1/2 and of the membrane-associated upstream protein kinases c-Src and PKCδ were significantly elevated, and increased migration rates, as assessed by in vitro wound-closure assays and time-lapse microscopy, were observed. Forced expression of RACK1, which is the plectin-binding receptor protein for activated PKCδ, in wild-type keratinocytes elevated their migration potential close to that of plectin-null cells. These data establish a link between cytolinker-controlled cytoarchitecture/scaffolding functions of keratin IFs and specific MAP kinase cascades mediating distinct cellular responses.

Cdk5 is a membrane-associated protein kinase whose mislocalization induces neuronal cell death

Cdk5 is a membrane-associated protein kinase whose mislocalization induces neuronal cell death

Agents that act by different mechanisms modulate the activity of protein kinase CβII isozyme in the rat spinal cord during peripheral inflammation

Hyperalgesia following unilateral complete Freund’s adjuvant-induced inflammation was characterized by paw withdrawal latency to thermal stimulus. Paw withdrawal latencies were significantly shorter on the complete Freund’s adjuvant-treated paw than on the contralateral paw of the complete Freund’s adjuvant- and the sham-treated rats. Total cytosolic protein kinase C activity in the lumbar enlargement was unchanged on the sides of the spinal cord ipsi- and contra-lateral to the inflamed paw. Membrane-associated activities of protein kinase Cα, protein kinase CβI and protein kinase Cγ did not change significantly on the sides of the cord ipsi- and contra-lateral to the inflammation. However, membrane-associated activity of protein kinase CβII was increased in the cord section ipsilateral to the inflammation, suggesting that increased translocation/activation of protein kinase CβII is related to thermal hyperalgesia. Dextrorphan (an N-methyl- d-aspartate receptor antagonist), L-703,606 (an NK-1 receptor antagonist) and an antisense oligodeoxynucleotide for a selective knockdown of protein kinase Cβ, reduced complete Freund’s adjuvant-induced hyperalgesia, and reversed significant changes in the membrane activity of protein kinase CβII on the spinal cord section ipsilateral to the inflamed paw. Dextrorphan and protein kinase Cβ antisense oligodeoxynucleotide were effective in reversing complete Freund’s adjuvant-induced increase in the activity of protein kinase CβII ipsilateral to the inflammation at all the doses tested, but L-703,606 was effective only at the highest dose. Furthermore, in the presence of inflammatory stimulus, dextrorphan and L-703,606 did not alter the activities of membrane-associated protein kinase Cα, protein kinase CβI, and protein kinase Cγ in the section of the spinal cord ipsi- and contra-lateral to the inflammation. Protein kinase Cβ antisense oligodeoxynucleotide had no significant effect on the membrane-associated activities of protein kinase Cα and protein kinase Cγ, but decreased the activities of both protein kinase CβI and protein kinase CβII and the expression of protein kinase Cβ isozyme in the spinal cord. The data provide evidence that a common molecular event that converges to initiate and maintain hyperalgesia may include the translocation and activation of protein kinase CβII in the spinal dorsal horn.

Structure and Mode of Action of the Membrane-permeabilizing Antimicrobial Peptide Pheromone Plantaricin A

The three-dimensional structure in dodecyl phosphocholine micelles of the 26-mer membrane-permeabilizing bacteriocin-like pheromone plantaricin A (PlnA) has been determined by use of nuclear magnetic resonance spectroscopy. The peptide was unstructured in water but became partly structured upon exposure to micelles. An amphiphilic alpha-helix stretching from residue 12 to 21 (possibly also including residues 22 and 23) was then formed in the C-terminal part of the peptide, whereas the N-terminal part remained largely unstructured. PlnA exerted its membrane-permeabilizing antimicrobial activity through a nonchiral interaction with the target cell membrane because the d-enantiomeric form had the same activity as the natural l-form. This nonchiral interaction involved the amphiphilic alpha-helical region in the C-terminal half of PlnA because a 17-mer fragment that contains the amphiphilic alpha-helical part of the peptide had antimicrobial potency that was similar to that of the l- and d-enantiomeric forms of PlnA. Also the pheromone activity of PlnA depended on this nonchiral interaction because both the l- and d-enantiomeric forms of the 17-mer fragment inhibited the pheromone activity. The pheromone activity also involved, however, a chiral interaction between the N-terminal part of PlnA and its receptor because high concentrations of the l-form (but not the d-form) of a 5-mer fragment derived from the N-terminal part of PlnA had pheromone activity. The results thus reveal a novel mechanism whereby peptide pheromones such as PlnA may function. An initial nonchiral interaction with membrane lipids induces alpha-helical structuring in a segment of the peptide pheromone. The peptide becomes thereby sufficiently structured and properly positioned in the membrane interface, thus enabling it to engage in a chiral interaction with its receptor in or near the membrane water interface. This membrane-interacting mode of action explains why some peptide pheromones/hormones such as PlnA sometimes display antimicrobial activity in addition to their pheromone activity.

In vitro and in silico analysis of signal peptides from the human blood fluke, Schistosoma mansoni

Proteins secreted by and anchored on the surfaces of parasites are in intimate contact with host tissues. The transcriptome of infective cercariae of the blood fluke, Schistosoma mansoni, was screened using signal sequence trap to isolate cDNAs encoding predicted proteins with an N-terminal signal peptide. Twenty cDNA fragments were identified, most of which contained predicted signal peptides or transmembrane regions, including a novel putative seven-transmembrane receptor and a membrane-associated mitogen-activated protein kinase. The developmental expression pattern within different life-cycle stages ranged from ubiquitous to a transcript that was highly upregulated in the cercaria. A bioinformatics-based comparison of 100 signal peptides from each of schistosomes, humans, a parasitic nematode and Escherichia coli showed that differences in the sequence composition of signal peptides, notably the residues flanking the predicted cleavage site, might account for the negative bias exhibited in the processing of schistosome signal peptides in mammalian cells.